The pleiotropic actions of insulin are initiated by binding of the hormone to its cell-surface receptor. This results in activation of the intracellular tyrosine kinase domain of the insulin receptor (IR) beta-subunit with subsequent phosphorylation of cellular substrates on tyrosine. A number of adaptor and effector proteins have been discovered that interact with the receptor beta-subunit and participate in the mediation of insulin signaling. Our laboratory has recently shown that the cytoplasmic domain of the IR contains a reactive cysteine thiol that can form a covalent complex with a thiol-reactive membrane-associated protein (TRAP) upon treatment of a number of cell types with bis-maleimidohexane, an irreversible homobifunctional crosslinking reagent with specificity for thiols. We have purified the IR-TRAP complex by an affinity-chromatography based approach using immobilized monoclonal IR antibodies followed by trypsin digestion of the purified protein band. Trypsin digests were then subjected to MALDI-TOF analyses to allow the identification of TRAP as phosphoinositide-specific phospholipase C-gamma 1 (PLCgamma1), an enzyme that plays a pivotal role in transmembrane signaling. The IR-PLCgamma1 complex is highly compartmentalized and is found concentrated in lipid rafts, unique cell-surface membrane microdomains with diverse roles in cellular physiology. Although recompartmentalization of PLCgamma1 to the lipid rafts controls its activation status, the molecular mechanism(s) by which it is recruited to the IR for proper insulin signaling is not understood. Molecular, biochemical and cellular techniques are currently used to elucidate this important problem. Insulin stimulation often leads to rapid reorganization of the actin cytoskeleton to generate the forces necessary for plasma membrane ruffling formation and a host of other cellular processes, including the redistribution of insulin-regulatable glucose transporter 4 in the cell surface. The stabilization of actin network and its attachment to cellular membranes is orchestrated by actin binding proteins. One group of these proteins, called filamins, has been shown to bind with both actin filaments and a number of macromolecules, notably small GTPases, and raft-associated caveolin-1. The co-localization of filamin and resident raft proteins is of physiological importance and provides evidence for the organization and clustering of lipid rafts by the actin cytoskeleton. We hypothesized that the recruitment of PLCgamma1 to the IR may depend on insulin?s ability to modulate filamin signaling. The availability of human melanoma cell lines (M2) that have spontaneously lost expression of filamin A and a subline with stable expression of recombinant filamin A (M2A7) enabled us to assess the role of this actin-binding protein in the recruitment of PLCgamma1 to the IR upon insulin stimulation. Our preliminary experiments have shown that the extent of IR-PLCgamma1 association elicited by insulin was markedly reduced in filamin-repleted cells when compared to cells lacking filamin A. In contrast, insulin yielded comparable activation of the IR autophosphorylation in both lines. Additional experiments revealed that filamin was constitutively present in IR immunoprecipitates and that the avidity of filamin for the IR was decreased after treatment of various cell types with insulin. Work is underway to further characterize the binding of filamin A to the IR and to its impact on insulin signaling. Insulin serves as an important signal for the maintenance of systemic glucose homeostasis under physiological conditions. The molecular basis for the development of insulin resistance in aging, obesity and in disease states such as type 2 diabetes are complex and remain elusive. Therefore, research to improve our understanding of IR-PLCgamma1 and IR-filamin interaction may offer novel targets for the search of the mechanism(s) of insulin resistance and may provide new insight into insulin signal transduction with broad consequences in cell biology.